Cystic fibrosis is a highly variable disorder of fluid and electrolyte transport that primarily affects the lung, pancreas, intestine, male reproductive tract and the sweat gland. Positional cloning identified CF Transmembrane conductance Regulator (CFTR) as the gene responsible for CF. It has been demonstrated that normal function of CFTR requires 1) efficient folding in the endoplasmic reticulum, 2) targeting to apical membranes in epithelial cells, 3) activation by b-adrenergic cAMP-dependent pathways, 4) generation of endogenous CI- currents and 5) regulation of separate sodium and chloride channels (and possibly other channels and transporters). The vexing question has been which process, or combination of processes, when altered by mutation in CFTR correlates with the severity of disease? We and others have shown that mutations found in patients can affect each of these processes, and that alteration in the maturation (#1) and chloride transport processes (#4) of CFTR partially correlate with disease severity. During the prior phase of this grant, we demonstrated that alteration in the regulatory function of CFTR (#5) correlates with disease severity, but only for some mutations. Thus, the mechanism by which certain mutations cause disease is unknown. The overall goal of this grant is to increase our understanding of CF pathophysiology by investigating the contribution of localization defects (#2) and altered cAMP-activation (#3) to the development of the CF phenotype. This will be achieved by pursuit of the following aims: 1) To identify proteins interacting with the C-terminus that participate in CFTR localization to apical membranes of polarized epithelial cells. 2) To determine whether mutations in regions other than the C-terminus affect CFTR localization and correlate with disease severity. 3) To determine whether defects in components of the cAMP-activation pathway alters CFTR function and creates a CF phenotype.